The proposed studies will concentrate on understanding the biochemistry of both normal and mutant human fructose diphosphate aldolase isozymes. These enzymes function in glycolysis and gluconeogenesis. The studies proposed will lead to a greater understanding of metabolism and hereditary human disease. The human disease, hereditary fructose intolerance (HFI), is a digestive disorder involving carbohydrate metabolism in the liver. Studies on the genes for these isozymes will eventually lead to a better understanding of enzyme mechanism, protein structure, protein folding, evolution, and the control of gene expression during tissue specific development. The scope of the initial proposal takes advantages of a set of human mutations which lead to the aforementioned digestive disease (HFI). Recombinant DNA technology will be employed for investigation of the precise nature of the mutations which manifest themselves in this disease. The mutations which lead to this disease will be identified and characterized. These studies will lead to methods of carrier detection and prenatal diagnosis in families with a history of the disease. The experimental approach will study the genetics of the disease in a classical sense as well as use the more modern approach of reverse genetics. The specific aims involve the isolation and characterization of the complete set of human genes encoding the aldolases. Then the mutant genes from patients with the HFI disease will be isolated and characterized. Genomic libraries will be constructed and screened for clones harboring these genes. The DNA sequence of the genes will be determined. In vitro transcription experiments will determine the flanking regions necessary for the promoter activities. The genes from HFI patients will be compared to the normal genes. The DNA will be from patients confirmed for the disease by biopsy and/or family analysis. They will be asked to participate by informed consent to provide blood samples. The mutations found will be analyzed be reverse genetics for their effect on the gene expression or enzyme activity. The normal gene will be changed to mimic the HFI mutant gene by in vitro mutagenesis. Finally, competition experiments performed in vivo will investigate the elements and modes of trans-regulatory control involved in this family of genes.